The ability to sense and adapt to new environments is required for fungal survival and pathogenesis. Changes in the level of extracellular amino acids may signal a 'new'environment, which results in induction of specific signaling pathways and acquisition of nutrients. The Ssy1-Ptr3-Ssy5 (SPS) amino acid sensing pathway is known to be involved in this environmental adaptation. The long-term objective of this proposal is to understand the molecular mechanisms underlying SPS signaling in eukaryotic cells using Saccharomyces cerevisiae as a model organism. In the presence of extracellular amino acids, Ssy5-dependent endoproteolytic processing of two transcription factors Stp1 and Stp2 leads to expression of amino acid transporters that facilitate amino acid uptake. The SPS amino acid sensing pathway exists in several pathogenic Candida species and is required for pathogenicity of C. albicans in an animal model. Our preliminary data have revealed unexpected activation of SPS-target gene expression under glucose limitation conditions that requires Gcn2, a kinase key to activation of the General Amino Acid Control (GAAC) response in cells deprived of amino acids, but not Gcn4, a transcription factor required for the GAAC. In cells experiencing amino acid starvation,Gcn2 increases the translation of GCN4 mRNA via an unusual mechanism involving small upstream open reading frames (uORF) in the leader sequence of GCN4 mRNA. This unique protein translational control is conserved in mammalian cells, with ATF4/5 as the counterpart of yeast GCN4. Although uORFs are predicted to exist in 10-25% eukaryotic genes, only a few uORFs have been studied in detail. Herein, we describe specific aims that we will use to study this newly discovered link between extracellular and intracellular amino acid sensing pathways through the common regulator Gcn2 using yeast genetics, cell biology and biochemical tools. We will also characterize the mechanism underlying Ubc4/5-dependent degradation of processed Stp2 and the role of Stp2 degradation in pathway regulation. These studies will provide novel insights into mechanisms underlying amino acid sensing and signaling in yeast.